Recombinant technology has been used to clone, express and purify several proteins of therapeutic or other economic value. Use of Eukaryotic host cells to express large quantity of recombinant proteins in large amounts has become increasingly important because of the ability of Eukaryotic cells to express the protein of interest in desired conformation. Method to improve protein expression often includes increasing gene dosage or copies, or by adding genetic elements that prolong life of cells. Various factors affect the ability of the expression vector to express the protein during fermentation. These include selection marker gene used for selection of vector containing cells from other cells, Orientation of the genes coding for product of interest, strength of the promoter linked to the gene for protein of the interest, the sequence of the 5′ untranslated and the translation initiation region, the efficiency of the 3′ untranslated region to polyadenylate and terminate transcription, the insertion site of the randomly integrated recombinant gene in the host chromosome, and the number of integrated copies of the gene that is being expressed. In spite of the plethora of available vectors, production of robust cell lines producing the polypeptide/protein at high concentration in a consistent manner is still challenging. Several other factors can influence the yield for expression of recombinant protein in mammalian cells, some of them are protein to be expressed, Media components, Host cell lines used, etc.
Other problem associated with industrial production of recombinant proteins using eukaryotic cells is related to stress conditions incurred by cells during the late stages of fermentation. During the late phases of the fermentation the number of cells are more as compared to early phases of fermentation. Hence, the protein of interest is produced at much faster rate. During the late phase of the fermentation, the cells are exposed to stress conditions such as high temperature, high osmotic pressure, metabolic inhibition, presence of heavy metals, viral infection, etc. These factors negatively affect the fermentation process and expression of the protein of interest by increasing the rate of cell death. The increase in cell death results in decrease in time of the fermentation cycles per batch. This result in decrease in overall yield of the protein produced per batch. This leads to increases in duration of the fermentation cycle to improve the overall yield of protein of interest and thus increasing the cost of production.
The patent application U.S. Pat. No. 7,935,808 discloses REVE sequences which may comprise one or more matrix attachment region (MAR) sequences. MAR sequences may occur in clusters within a rEVE sequence, including in clusters at the 5′ and/or 3′ terminal regions of a rEVE sequence. It further discloses Dihyrofolate reductase for higher survivability and/or higher growth rate. The patent discloses use of Heat shock protein (HSP) with other elements like MAR, gene of interest to achieve stable cell. However, the incorporation of three genes i.e. HSP gene, MAR sequence and Gene for protein, into its genome at a stable location is not disclosed.
Despite significant progress in improving the yield from these cells, the process to the selection, identification, and maintenance of high-producing cell lines remains cumbersome, time consuming, and often of uncertain outcome. Thus, there is a need in the art to design improved expression vectors useful for protein expression in mammalian cells which can overcome the deficiencies of the known methods and thus improve the expression of the vectors to yield highly stable and viable cell lines. In the present invention, the vectors so designed will provide an efficient generation of stable cell lines expressing the product of interest at desired levels. The vector generates a high expression stable cells lines with higher viability and stability and yet reduces the total time of fermentation by reducing the number of fermentation cycles and overcomes the drawbacks presented by the prior art.